Previous studies in the Howell lab have found that addition of neutral osmolytes to an R-plasmid encoded dihydrofolate reductase (R67 DHFR) as well as the non-homologous E. coli chromosomal DHFR (EcDHFR) result in weaker binding of the substrate, dihydrofolate (DHF). This result contrasts with the tighter binding of NADPH under these same conditions. The cofactor results are more typical, as the decreased water content reduces the desolvation penalty associated with binding. A model to explain the weaker binding of DHF to DHFR hypothesizes that weak interactions occur between dihydrofolate and various osmolytes. If the DHFosmolyte interaction is stronger than the DHFwater interaction, then it will take more energy to lose the DHFosmolyte pair and binding of DHF to DHFR will be more difficult. (In other words, water prefers to interact with osmolytes as compared to folate.) To expand this solvent substitution hypothesis of enzyme action, different folate derivatives as well as different folate utilizing enzymes will be studied. Aim 1 proposes a series of experiments to study how DHF and folate derivatives interact with osmolytes. A Hansch plot will be used to determine if hydrophobicity is the signature element that makes folate sticky. Aim 2 extends these biochemical studies to include other enzymes in one carbon metabolism. Aim 3 returns to DHFR to study the effects of macromolecular crowding on DHFR activity as the motifs found in osmolytes can also be found in proteins, leading to the proposal that DHFcrowder interactions will occur and affect catalytic efficiency. Aim 4 proposes a series of in vivo tests of the folateosmolyte interaction model by genetic complementation assays performed under low water activity conditions in E. coli. Osmotic stress is predicted to result in lower catalytic efficiencies for enzymes involved in folate mediated one carbon metabolism, ultimately leading to blockage of growth. Aim 4B considers osmolyte effects on antibacterial resistance conferred by DHFR upon host E. coli and addresses what happens to the DHF and DHFR concentrations. This groundbreaking research identifies osmolality (or low water activity) as a key environmental factor involved in modulating DHFR function. By extension, other important folate utilizing enzymes will also be impacted. Factors that affect the folate, or vitamin B9, concentration, half-life and accessibility are important to human health as the redox forms of folate are substrates and coenzymes in the one carbon cycle, which impacts amino acid and nucleic acid metabolism. As animals do not synthesize folate or vitamin B9, they must obtain it from their diet. The consequences of insufficient folate nutrition can be seen in the adverse outcomes of pregnancy, thus the recent folate fortification of foods. Antifolates are also used as treatments o bacterial infections, malaria, cancer, arthritis, cardiovascular disease, etc. These are a few examples where folate biochemistry plays a role in human health.